MIT biological engineers have developed a modular system of proteins that can detect a particular DNA sequence in a cell and then trigger a specific response, such as cell death.

This system can be customized to detect any DNA sequence in a mammalian cell and then trigger a desired response, including killing cancer cells or cells infected with a virus, the researchers say.

"There is a range of applications for which this could be important," says James Collins, the Termeer Professor of Medical Engineering and Science in MIT's Department of Biological Engineering and Institute of Medical Engineering and Science (IMES). "This allows you to readily design constructs that enable a programmed cell to both detect DNA and act on that detection, with a report system and/or a respond system."

Collins is the senior author of a Sept. 21 Nature Methods paper describing the technology, which is based on a type of DNA-binding proteins known as zinc fingers. These proteins can be designed to recognize any DNA sequence.

"The technologies are out there to engineer proteins to bind to virtually any DNA sequence that you want," says Shimyn Slomovic, an IMES postdoc and the paper's lead author. "This is used in many ways, but not so much for detection. We felt that there was a lot of potential in harnessing this designable DNA-binding technology for detection."

Sense and respond

To create their new system, the researchers needed to link zinc fingers' DNA-binding capability with a consequence -- either turning on a fluorescent protein to reveal that the target DNA is present or generating another type of action inside the cell.

The researchers achieved this by exploiting a type of protein known as an "intein" -- a short protein that can be inserted into a larger protein, splitting it into two pieces. The split protein pieces, known as "exteins," only become functional once the intein removes itself while rejoining the two halves.

Collins and Slomovic decided to divide an intein in two and then attach each portion to a split extein half and a zinc finger protein. The zinc finger proteins are engineered to recognize adjacent DNA sequences within the targeted gene, so if they both find their sequences, the inteins line up and are then cut out, allowing the extein halves to rejoin and form a functional protein. The extein protein is a transcription factor designed to turn on any gene the researchers want.

In this paper, they linked green fluorescent protein (GFP) production to the zinc fingers' recognition of a DNA sequence from an adenovirus, so that any cell infected with this virus would glow green.

This approach could be used not only to reveal infected cells, but also to kill them. To achieve this, the researchers could program the system to produce proteins that alert immune cells to fight the infection, instead of GFP.

"Since this is modular, you can potentially evoke any response that you want," Slomovic says. "You could program the cell to kill itself, or to secrete proteins that would allow the immune system to identify it as an enemy cell so the immune system would take care of it."

The MIT researchers also deployed this system to kill cells by linking detection of the DNA target to production of an enzyme called NTR. This enzyme activates a harmless drug precursor called CB 1954, which the researchers added to the petri dish where the cells were growing. When activated by NTR, CB 1954 kills the cells.

Future versions of the system could be designed to bind to DNA sequences found in cancerous genes and then produce transcription factors that would activate the cells' own programmed cell death pathways.

Research tool

The researchers are now adapting this system to detect latent HIV proviruses, which remain dormant in some infected cells even after treatment. Learning more about such viruses could help scientists find ways to permanently eliminate them.

"Latent HIV provirus is pretty much the final barrier to curing AIDS, which currently is incurable simply because the provirus sequence is there, dormant, and there aren't any ways to eradicate it," Slomovic says.

While treating diseases using this system is likely many years away, it could be used much sooner as a research tool, Collins says. For example, scientists could use it to test whether genetic material has been successfully delivered to cells that scientists are trying to genetically alter. Cells that did not receive the new gene could be induced to undergo cell death, creating a pure population of the desired cells.

It could also be used to study chromosomal inversions and transpositions that occur in cancer cells, or to study the 3-D structure of normal chromosomes by testing whether two genes located far from each other on a chromosome fold in such a way that they end up next to each other, the researchers say.

— Scientists at UC San Diego, UC Santa Barbara and MIT have developed nanometer-sized “nanoworms” that can cruise through the bloodstream without significant interference from the body’s immune defense system and—like tiny anti-cancer missiles—home in on tumors.

Their discovery, detailed in this week’s issue of the journal Advanced Materials, is reminiscent of the 1966 science fiction movie, the Fantastic Voyage, in which a submarine is shrunken to microscopic dimensions, then injected into the bloodstream to remove a blood clot from a diplomat’s brain.

Using nanoworms, doctors should eventually be able to target and reveal the location of developing tumors that are too small to detect by conventional methods. Carrying payloads targeted to specific features on tumors, these microscopic vehicles could also one day provide the means to more effectively deliver toxic anti-cancer drugs to these tumors in high concentrations without negatively impacting other parts of the body.

“Most nanoparticles are recognized by the body's protective mechanisms, which capture and remove them from the bloodstream within a few minutes,” said Michael Sailor, a professor of chemistry and biochemistry at UC San Diego who headed the research team. “The reason these worms work so well is due to a combination of their shape and to a polymer coating on their surfaces that allows the nanoworms to evade these natural elimination processes. As a result, our nanoworms can circulate in the body of a mouse for many hours.”

“When attached to drugs, these nanoworms could offer physicians the ability to increase the efficacy of drugs by allowing them to deliver them directly to the tumors,” said Sangeeta Bhatia, a physician, bioengineer and a professor of Health Sciences and Technology at MIT who was part of the team. “They could decrease the side effects of toxic anti-cancer drugs by limiting their exposure of normal tissues and provide a better diagnosis of tumors and abnormal lymph nodes.”

The scientists constructed their nanoworms from spherical iron oxide nanoparticles that join together, like segments of an earthworm, to produce tiny gummy worm-like structures about 30 nanometers long—or about 3 million times smaller than an earthworm. Their iron-oxide composition allows the nanoworms to show up brightly in diagnostic devices, specifically the MRI, or magnetic resonance imaging, machines that are used to find tumors.

“The iron oxide used in the nanoworms has a property of superparamagnetism, which makes them show up very brightly in MRI,” said Sailor. “The magnetism of the individual iron oxide segments, typically eight per nanoworm, combine to provide a much larger signal than can be observed if the segments are separated. This translates to a better ability to see smaller tumors, hopefully enabling physicians to make their diagnosis of cancer at earlier stages of development.”

In addition to the polymer coating, which is derived from the biopolymer dextran, the scientists coated their nanoworms with a tumor-specific targeting molecule, a peptide called F3, developed in the laboratory of Erkki Ruoslahti, a cell biologist and professor at the Burnham Institute for Medical Research at UC Santa Barbara. This peptide allows the nanoworms to target and home in on tumors.

“Because of its elongated shape, the nanoworm can carry many F3 molecules that can simultaneously bind to the tumor surface,” said Sailor. “And this cooperative effect significantly improves the ability of the nanoworm to attach to a tumor.”

The scientists were able to verify in their experiments that their nanoworms homed in on tumor sites by injecting them into the bloodstream of mice with tumors and following the aggregation of the nanoworms on the tumors. They found that the nanoworms, unlike the spherical nanoparticles of similar size that were shuttled out of the blood by the immune system, remained in the bloodstream for hours.

“This is an important property because the longer these nanoworms can stay in the bloodstream, the more chances they have to hit their targets, the tumors,” said Ji-Ho Park, a UC San Diego graduate student in materials science and engineering working in Sailor’s laboratory.

Park was the motivating force behind the discovery when he found by accident that the gummy worm aggregates of nanoparticles stayed for hours in the bloodstream despite their relatively large size.While it’s not clear yet to the researchers why, Park notes that “the nanoworm’s flexibly moving, one dimensional structure may be one the reasons for its long life in the bloodstream.”

The researchers are now working on developing ways to attach drugs to the nanoworms and chemically treating their exteriors with specific chemical “zip codes,” that will allow them to be delivered to specific tumors, organs and other sites in the body.

“We are now using nanoworms to construct the next generation of smart tumor-targeting nanodevices,” said Ruoslahti. We hope that these devices will improve the diagnostic imaging of cancer and allow pinpoint targeting of treatments into cancerous tumors.”